During the past year we completed a series of experiments comparing testosterone secretion by rat, rabbit, dog, guinea pig and hamster testes perfused in vitro. Species differences in testosterone secretion were discovered. These species differences in testosterone secretion were found not to be correlated with the mass of Leydig cells present in the testis. Instead, testosterone secretion was correlated directly with the amount of smooth endoplasmic reticulum in Leydig cells. Testosterone secretion and the proportion of Leydig cell smooth endoplasmic reticulum was highest in the guinea pig followed in order by rabbit, dog, rat and hamster. Finally, it was shown that the flux of steroids through the testosterone biosynthetic pathway was markedly different in rats and rabbits. Apparently, rabbit testes perfused in vitro synthesized testosterone from pregnenolone via 17 hydroxypregnenolone yields dehydroepiandrosterone yields delta 5 androstenediol yields testosterone. By contrast, two pathways were found to predominate in rat testes perfused in vitro. Pregnenolone yields 17 hydroxypregnenolone yields 17 hydroxyprogesterone yields delta 4 androstenedione yields testosterone and pregnenolone yields progesterone yields 17 hydroxyprogesterone yields delta 4 androstenedione yields testosterone.